We have made an anti-mesothelin immunotoxin in which the major human B cell epitopes have been silenced or removed and are evaluating it in humans with cancer. We have also identified the major T cell epitopes in PE38 and used this data to construct LMB-20, an immunotoxin with T cell and B cell epitopes suppressed. LMB-20 is very active in killing mesothelin-expressing cancer cells and has good anti-tumor activity in mice. We have found that immunotoxins synergize several commonly used anti-cancer agents: Actinomycin D, Taxanes, Panbinostatat and Azacytidine to kill cancer cells in culture to cause regressions of tumors in mice and to prevent the development of drug resistance. To increase immunotoxin half-life we have introduced cys residues into non-essential positions in the toxin and used these residues to add PEG molecules of different sizes to the toxin. We evaluated these for cytotoxic activity in cell culture and in mice with tumors. Because we observed that some patients treated with immunotoxin SS1P, developed anti-tumor immunity, we have developed a syngeneic mouse tumor model to study the interactions of the immune system with immunotoxin therapy. We have found that anti-mesothelin immunotoxins synergize with anti-CTLA-4 to cause tumor regressions and anti-tumor immunity in mice. To develop a new treatment for multiple myeloma, we have made two new immunotoxins targeting BCMA, which is expressed on almost all myelomas and we have shown these are very active in killing myeloma cells in culture and in mice bearing myeloma tumors. Finally, because the development of anti-drug antibodies interferes with the activity of immunotoxins in humans, we are collaborating with Selecta Biosciences and have shown that ADA formation vs immunotoxins is prevented and immune tolerance induced by co-administration nanoparticles containing rapamycin and immunotoxin. A clinical trial combining these two agents is planned to open this year.